Project Summary/Abstract Ewing sarcoma, the second most common pediatric bone cancer, represents a significant therapeutic challenge. Current treatment protocols, which include chemotherapy, surgery and radiotherapy, achieve event free survival rates of 70% for patients with localized disease. Patients with metastasis have survival rates of only 30% despite intensified therapy regimens. There is thus a pressing need to identify new therapeutic targets for this disease. Molecularly, Ewing sarcoma is characterized by translocations between the EWS gene and ETS transcription factors, most commonly FLI1 (in 85% of cases). These events produce the EWS-FLI1 fusion protein, which is the main oncogenic driver of Ewing sarcoma and has been shown to encode an aberrant transcriptional regulator. Our laboratory and others have recently used genome-wide chromatin profiling to determine that EWS-FLI1 orchestrates a tumor specific gene regulation program primarily by altering chromatin states at its binding sites. These studies have shown that EWS-FLI1 can either activate or repress chromatin depending on the underlying DNA sequence. At GGAA repeats, EWS-FLI1 functions as a ?pioneer? factor that can activate a large set of enhancers starting from closed chromatin. At canonical non- repeat ETS sites, EWS-FLI1 binding leads to displacement of wildtype ETS factors and repression. We hypothesize that EWS-FLI1 interacts with a network of cooperating proteins that enable its chromatin regulation activities and that these proteins may represent important new therapeutic targets for Ewing sarcoma. We now propose to identify and characterize the EWS-FLI1 protein interaction network through a combination of mass spectrometry, chromatin profiling and functional studies. Preliminary results show interactions between EWS-FLI1 and MLL complexes and our studies will pursue the characterization of these highly relevant therapeutic targets in addition to other interacting proteins. Our aims are: 1) To define the EWS- FLI1 protein interaction network, 2) To determine the contribution of MLL complexes and other interacting proteins to EWS-FLI1 driven gene regulation, 3) To define key domains in MLL/SET proteins and other EWS- FLI1 interacting proteins that impact EWS-FLI1 function and Ewing sarcoma cell survival. Our project will also involve significant interactions with other Projects and Cores in the Center. We anticipate that our studies will have a major impact on our understanding of the mechanisms of action of EWS-FLI1 and will point to new therapeutic targets for Ewing sarcoma.